Although magnesium is the most abundant divalent metal ion found in most cells, the regulation of the cytosolic free magnesium ion concentration and the role which alterations in the level of this ion may play in mediating the pathology of a variety of toxins, is relatively unknown and unexplored. Until recently there were very few methods that were easy and reliable for measuring cytosolic free magnesium levels. As a result, very little is known about how the cell regulates free magnesium ion concentration, and whether free magnesium ion concentration is involved in regulating cell function. Two recent developments have made this an exciting new area of research. Our laboratory has recently introduced new and reliable NMR and fluorescent indicators for free magnesium ion concentration. Using these methods, our group has established that basal free magnesium ion concentration levels are in the range of 0.3 - 1.0 mM; values that are considerably lower than the 3-4 mM which had been suggested by most previous methods. This result, coupled with recent studies showing that as Mg is altered around the value of 0.5 mM it has a large effect on many cell processes such as K and Ca ion channel activity, Ca release from the sarcoplasmic reticulum and protein studies, provide strong support for an important regulatory role for free magnesium ion concentration. However, before we can address the role of free magnesium ion concentration in pathology, we need to have an understanding of how the cell regulates free magnesium ion concentration. We have determined that both H(+) and Ca(2+) compete with Mg(2+) for intracellular binding sites. Thus, a sustained increase in calcium ion concentration or a decrease in pH causes a secondary increase in the free magnesium ion concentration, and since this increase in Magnesium ion concentration, occurs in Mg-free solution, it does not require Mg transport across the plasma membrane. The cell maintains intracellular free Mg(2+) well below electrochemical equilibrium; Magnesium ion concentration would be 20 mM if it were in electrochemical equilibrium with an extracellular magnesium ion concentration of 1 mM and a membrane potential of -40 mV. Thus, the cell must have some transport mechanism for extruding Mg(2+). We are investigating the role of Na/Mg exchange. We are also investigating whether an alteration in Magnesium ion concentration accompanies changes in cell volume.